Semevolos SA, Marchant EA.The marginal transitional zone (MTZ) is peripherally located within the diarthrodial joint, and represents the junction of synovium, fibrous joint capsule, articular cartilage, periosteum, and bone. The purpose of this study is to characterize age-related differences in protein expression of matrix and molecular regulators in the marginal transitional zone of neonatal and weanling foals. Several families of proteins with known roles in cartilage and bone development are investigated, including matrix molecules, members of the Wnt signaling family, apoptotic factors and paracrine cell signaling...
Singh UK, Patel R.In vitro refolding of denatured protein and the influence of the alkyl chain on the refolding of a protein were tested using long chain imidazolium chloride salts, 1-methyl-3-octylimidazolium chloride [Cmim][Cl], and 1-decyl-3-methylimidazolium chloride [Cmim][Cl]. The horse heart cytochrome c (h-cyt c) was denatured by urea and guanidinium hydrochloride (GdnHCl), as well as by base-induced denaturation at pH 13, to provide a broad overview of the overall refolding behavior. The variation in the alkyl chain of the ionic liquids (ILs) showed a profound effect on the refolding of denatured h-cyt...
De Wael K, De Belder S, Van Vlierberghe S, Van Steenberge G, Dubruel P, Adriaens A.The aim of this paper is to emphasize the strength of gelatin as a stable matrix for redox enzymes. Cyclic voltammetry has been applied for a detailed electrochemical study of horse heart cytochrome c (HHC) entrapped in a gelatin matrix immobilized on a gold electrode. The influence of the HHC concentration, the mass percentage of the gelatin and the nature of the gelatin on the electrochemical behaviour of HHC have been described in detail. In addition, attenuated total reflection infrared (ATR-IR) spectroscopy was used to prove the immobilization on a qualitative and conformational level. Th...
Gorgoglione V, Laraspata D, La Piana G, Marzulli D, Lofrumento NE.The data reported are fully consistent with the well-known observation that exogenous cytochrome c (cyto-c) molecules do not permeate through the outer membrane of mitochondria (MOM) incubated in isotonic medium (250 mM sucrose). Cyto-c is unable to accept electrons from the sulfite/cyto-c oxido-reductase (Sox) present in the intermembrane space, unless mitochondria are solubilized. Mitochondria incubated in a very high hypotonic medium (25 mM sucrose), in contrast to any expectation, continue to be not permeable to added cyto-c even if Sox and adenylate kinase are released into the medium. Th...
Schejter A, Ryan MD, Blizzard ER, Zhang C, Margoliash E, Feinberg BA.Contrary to most heme proteins, ferrous cytochrome c does not bind ligands such as cyanide and CO. In order to quantify this observation, the redox potential of the ferric/ferrous cytochrome c-cyanide redox couple was determined for the first time by cyclic voltammetry. Its E0' was -240 mV versus SHE, equivalent to -23.2 kJ/mol. The entropy of reaction for the reduction of the cyanide complex was also determined. From a thermodynamic cycle that included this new value for the cyt c cyanide complex E0', the binding constant of cyanide to the reduced protein was estimated to be 4.7 x 10(-3) L M(...
Rasmussen T, Brittain T, Berks BC, Watmough NJ, Thomson AJ.Nitrous oxide reductase (N2OR) catalyses the final step of bacterial denitrification, the two-electron reduction of nitrous oxide (N2O) to dinitrogen (N2). N2OR contains two metal centers; a binuclear copper center, CuA, that serves to receive electrons from soluble donors, and a tetranuclear copper-sulfide center, CuZ, at the active site. Stopped flow experiments at low ionic strengths reveal rapid electron transfer (kobs=150 s-1) between reduced horse heart (HH) cytochrome c and the CuA center in fully oxidized N2OR. When fully reduced N2OR was mixed with oxidized cytochrome c, a similar rat...
Yamada S, Suruga K, Ogawa M, Hama T, Satoh T, Kawachi R, Nishio T, Oku T.The appearance of NO2- reducing activity of cytochrome c (Cyt c) upon heat denaturation was investigated with equine heart Cyt c. Denatured equine heart Cyt c (dCyt c), which was treated at 100 degrees C for 30 min, had NO2- reducing activity in the presence of dithionite and methylviologen in an aqueous solution under anaerobic conditions. In contrast, hemoglobin and myoglobin had no such activity under the same conditions. Using spectroscopic methods, we found that the appearance of this activity in the Cyt c was due to the following intramolecular changes: unfolding of the peptide chain, ex...
Paynter S, Russell DA.Recently, the observation of pH-induced conformational changes of biomolecules supported on carboxymethyldextran (CMD)-coated surfaces measured using surface plasmon resonance (SPR) has been reported. However, it is apparent that the evidence reported in the literature is ambiguous. The research presented in this paper describes investigations to study the changing SPR signal of immobilized biomolecules as a function of varying pH, to provide a detailed understanding of the origin of the pH-induced changes in the SPR profile. SPR measurements were performed with cytochrome c, concanavalin A, a...
Salamon Z, Tollin G.Cyclic voltammetry has been used to study the effects of interactions between horse cytochrome c and solid-supported planar lipid membranes, comprised of either egg phosphatidylcholine (PC) or PC plus 20 mol.% cardiolipin (CL), on the redox potential and the electrochemical electron transfer rate between the protein and a semiconductor electrode. Experiments were performed over a wide range of cytochrome c concentrations (0-440 microM) at low (20 mM) and medium (160 mM) ionic strengths. Three types of electrochemical behavior were observed, which varied as a function of the experimental condit...
Science (New York, N.Y.)December 11, 1992
Volume 258, Issue 5089 1748-1755 doi: 10.1126/science.1334573
Pelletier H, Kraut J.The crystal structure of a 1:1 complex between yeast cytochrome c peroxidase and yeast iso-1-cytochrome c was determined at 2.3 A resolution. This structure reveals a possible electron transfer pathway unlike any previously proposed for this extensively studied redox pair. The shortest straight line between the two hemes closely follows the peroxidase backbone chain of residues Ala194, Ala193, Gly192, and finally Trp191, the indole ring of which is perpendicular to, and in van der Waals contact with, the peroxidase heme. The crystal structure at 2.8 A of a complex between yeast cytochrome c pe...
Di Bello C, Vita C, Gozzini L.A strategy based on complexation-assisted condensation of large synthetic protein fragments and mitochondria-mediated stereospecific heme insertion has been utilized to assemble a functional molecule corresponding to native horse heart holocytochrome c. This original approach offers the unique opportunity of selective modifications both in the C-terminal and in the N-terminal regions of the apoprotein and may represent an useful alternative to site-directed mutagenesis, particularly when D-amino acids, chemically and/or isotopically modified or other unnatural amino acids have to be introduced...
Gopal D, Wilson GS, Earl RA, Cusanovich MA.The ion binding properties of horse, bovine, and tuna cytochrome c (both oxidized and reduced) have been measured using a combination of ultrafiltration, neutron activation, and ion chromatography. The ions investigated were chloride, phosphate, and Tris-cacodylate. Ion chromatography and neutron activation analysis techniques were employed to determine the concentration of free anions. Binding constants are obtained from modified Scatchard plots (in the range of 10-2000 M-1). The redox potentials for cytochrome c at different ionic strengths, pH 7.0, have been determined. In this paper we rep...
Vitello LB, Erman JE.The binding of horse heart cytochrome c to yeast cytochrome c peroxidase in which the heme group was replaced by protoporphyrin IX was determined by a fluorescence quenching technique. The association between ferricytochrome c and cytochrome c peroxidase was investigated at pH 6.0 in cacodylate/KNO3 buffers. Ionic strength was varied between 3.5 mM and 1.0 M. No binding occurs at 1.0 M ionic strength although there was a substantial decrease in fluorescence intensity due to the inner filter effect. After correcting for the inner filter effect, significant quenching of porphyrin cytochrome c pe...
Satterlee JD, Moench S.Proton hyperfine resonance assignments for cytochromes c from several species are currently being successfully pursued by several laboratories. These efforts focus mostly on the ferrous forms. In contrast to that work, we have pursued assignments of the proton hyperfine shifted resonances for horse and tuna ferricytochromes c. Our results indicate that assignments are nearly identical in those two proteins. Using the pre-steady state nuclear Overhauser effect, several additional assignments have been made for the tuna protein, whereas for the horse protein, the following protons have been assi...
Borin G, Filippi B, Cavaggion F, Marchiori F.A solution synthesis is described of the partially protected N alpha-benzyloxycarbonylheptadecapeptide Z-Lys (Tfa)-Thr-Glu-Arg-Glu-Asp-Leu-Ile-Ala-Tyr-Leu-Lys (Tfa)-Lys (Tfa)-Ala-Thr-Asn-Glu (OBu t)-OBu t corresponding to sequence 88-104 of horse heart cytochrome c. The synthesis is achieved through the preparation of two subunits H1 (sequence 88-96) and H2 (sequence 97-104) and their linkage by an azide coupling step.
Borin G, Filippi B, Stivanello D, Marchiori F.A solution synthesis of Z-Gly-Thr-Lys (Tfa)-Met-Ile-Phe-Ala-Gly-Ile-Lys (Tfa)-Lys (Tfa)-NHNH-Boc corresponding to the sequence 77-87 of horse heart cytochrome c is described. The protected undecapeptide was obtained from intermediate hepta- and tetrapeptide fragments by an azide coupling.
Warme PK, Scheraga HA.The reported X-ray structure of horse-heart ferricytochrome c has been refined by conformational energy calculations, using a three-stage computational procedure. In stage I, the atomic positions are adjusted to conform to idealized bond lengths and bond angles characteristic of small amino acid derivatives, while yet remaining as close as possible to the X-ray coordinates. In stage II, atomic overlaps are eliminated by adjusting the backbone and side-chain dihedral angles to minimize the nonbonded energy, hydrogen-bonded energy, and rotational energy contributions. In the final stage of refin...
Science (New York, N.Y.)December 11, 1992
Volume 258, Issue 5089 1748-1755 doi: 10.1126/science.1334573
Pelletier H, Kraut J.The crystal structure of a 1:1 complex between yeast cytochrome c peroxidase and yeast iso-1-cytochrome c was determined at 2.3 A resolution. This structure reveals a possible electron transfer pathway unlike any previously proposed for this extensively studied redox pair. The shortest straight line between the two hemes closely follows the peroxidase backbone chain of residues Ala194, Ala193, Gly192, and finally Trp191, the indole ring of which is perpendicular to, and in van der Waals contact with, the peroxidase heme. The crystal structure at 2.8 A of a complex between yeast cytochrome c pe...
Salamon Z, Tollin G.Cyclic voltammetry has been used to study the effects of interactions between horse cytochrome c and solid-supported planar lipid membranes, comprised of either egg phosphatidylcholine (PC) or PC plus 20 mol.% cardiolipin (CL), on the redox potential and the electrochemical electron transfer rate between the protein and a semiconductor electrode. Experiments were performed over a wide range of cytochrome c concentrations (0-440 microM) at low (20 mM) and medium (160 mM) ionic strengths. Three types of electrochemical behavior were observed, which varied as a function of the experimental condit...
Gopal D, Wilson GS, Earl RA, Cusanovich MA.The ion binding properties of horse, bovine, and tuna cytochrome c (both oxidized and reduced) have been measured using a combination of ultrafiltration, neutron activation, and ion chromatography. The ions investigated were chloride, phosphate, and Tris-cacodylate. Ion chromatography and neutron activation analysis techniques were employed to determine the concentration of free anions. Binding constants are obtained from modified Scatchard plots (in the range of 10-2000 M-1). The redox potentials for cytochrome c at different ionic strengths, pH 7.0, have been determined. In this paper we rep...
Rasmussen T, Brittain T, Berks BC, Watmough NJ, Thomson AJ.Nitrous oxide reductase (N2OR) catalyses the final step of bacterial denitrification, the two-electron reduction of nitrous oxide (N2O) to dinitrogen (N2). N2OR contains two metal centers; a binuclear copper center, CuA, that serves to receive electrons from soluble donors, and a tetranuclear copper-sulfide center, CuZ, at the active site. Stopped flow experiments at low ionic strengths reveal rapid electron transfer (kobs=150 s-1) between reduced horse heart (HH) cytochrome c and the CuA center in fully oxidized N2OR. When fully reduced N2OR was mixed with oxidized cytochrome c, a similar rat...
Paynter S, Russell DA.Recently, the observation of pH-induced conformational changes of biomolecules supported on carboxymethyldextran (CMD)-coated surfaces measured using surface plasmon resonance (SPR) has been reported. However, it is apparent that the evidence reported in the literature is ambiguous. The research presented in this paper describes investigations to study the changing SPR signal of immobilized biomolecules as a function of varying pH, to provide a detailed understanding of the origin of the pH-induced changes in the SPR profile. SPR measurements were performed with cytochrome c, concanavalin A, a...
De Wael K, De Belder S, Van Vlierberghe S, Van Steenberge G, Dubruel P, Adriaens A.The aim of this paper is to emphasize the strength of gelatin as a stable matrix for redox enzymes. Cyclic voltammetry has been applied for a detailed electrochemical study of horse heart cytochrome c (HHC) entrapped in a gelatin matrix immobilized on a gold electrode. The influence of the HHC concentration, the mass percentage of the gelatin and the nature of the gelatin on the electrochemical behaviour of HHC have been described in detail. In addition, attenuated total reflection infrared (ATR-IR) spectroscopy was used to prove the immobilization on a qualitative and conformational level. Th...
Schejter A, Ryan MD, Blizzard ER, Zhang C, Margoliash E, Feinberg BA.Contrary to most heme proteins, ferrous cytochrome c does not bind ligands such as cyanide and CO. In order to quantify this observation, the redox potential of the ferric/ferrous cytochrome c-cyanide redox couple was determined for the first time by cyclic voltammetry. Its E0' was -240 mV versus SHE, equivalent to -23.2 kJ/mol. The entropy of reaction for the reduction of the cyanide complex was also determined. From a thermodynamic cycle that included this new value for the cyt c cyanide complex E0', the binding constant of cyanide to the reduced protein was estimated to be 4.7 x 10(-3) L M(...
Singh UK, Patel R.In vitro refolding of denatured protein and the influence of the alkyl chain on the refolding of a protein were tested using long chain imidazolium chloride salts, 1-methyl-3-octylimidazolium chloride [Cmim][Cl], and 1-decyl-3-methylimidazolium chloride [Cmim][Cl]. The horse heart cytochrome c (h-cyt c) was denatured by urea and guanidinium hydrochloride (GdnHCl), as well as by base-induced denaturation at pH 13, to provide a broad overview of the overall refolding behavior. The variation in the alkyl chain of the ionic liquids (ILs) showed a profound effect on the refolding of denatured h-cyt...
Satterlee JD, Moench S.Proton hyperfine resonance assignments for cytochromes c from several species are currently being successfully pursued by several laboratories. These efforts focus mostly on the ferrous forms. In contrast to that work, we have pursued assignments of the proton hyperfine shifted resonances for horse and tuna ferricytochromes c. Our results indicate that assignments are nearly identical in those two proteins. Using the pre-steady state nuclear Overhauser effect, several additional assignments have been made for the tuna protein, whereas for the horse protein, the following protons have been assi...
Gorgoglione V, Laraspata D, La Piana G, Marzulli D, Lofrumento NE.The data reported are fully consistent with the well-known observation that exogenous cytochrome c (cyto-c) molecules do not permeate through the outer membrane of mitochondria (MOM) incubated in isotonic medium (250 mM sucrose). Cyto-c is unable to accept electrons from the sulfite/cyto-c oxido-reductase (Sox) present in the intermembrane space, unless mitochondria are solubilized. Mitochondria incubated in a very high hypotonic medium (25 mM sucrose), in contrast to any expectation, continue to be not permeable to added cyto-c even if Sox and adenylate kinase are released into the medium. Th...
Vitello LB, Erman JE.The binding of horse heart cytochrome c to yeast cytochrome c peroxidase in which the heme group was replaced by protoporphyrin IX was determined by a fluorescence quenching technique. The association between ferricytochrome c and cytochrome c peroxidase was investigated at pH 6.0 in cacodylate/KNO3 buffers. Ionic strength was varied between 3.5 mM and 1.0 M. No binding occurs at 1.0 M ionic strength although there was a substantial decrease in fluorescence intensity due to the inner filter effect. After correcting for the inner filter effect, significant quenching of porphyrin cytochrome c pe...
Warme PK, Scheraga HA.The reported X-ray structure of horse-heart ferricytochrome c has been refined by conformational energy calculations, using a three-stage computational procedure. In stage I, the atomic positions are adjusted to conform to idealized bond lengths and bond angles characteristic of small amino acid derivatives, while yet remaining as close as possible to the X-ray coordinates. In stage II, atomic overlaps are eliminated by adjusting the backbone and side-chain dihedral angles to minimize the nonbonded energy, hydrogen-bonded energy, and rotational energy contributions. In the final stage of refin...
Borin G, Filippi B, Cavaggion F, Marchiori F.A solution synthesis is described of the partially protected N alpha-benzyloxycarbonylheptadecapeptide Z-Lys (Tfa)-Thr-Glu-Arg-Glu-Asp-Leu-Ile-Ala-Tyr-Leu-Lys (Tfa)-Lys (Tfa)-Ala-Thr-Asn-Glu (OBu t)-OBu t corresponding to sequence 88-104 of horse heart cytochrome c. The synthesis is achieved through the preparation of two subunits H1 (sequence 88-96) and H2 (sequence 97-104) and their linkage by an azide coupling step.
Di Bello C, Vita C, Gozzini L.A strategy based on complexation-assisted condensation of large synthetic protein fragments and mitochondria-mediated stereospecific heme insertion has been utilized to assemble a functional molecule corresponding to native horse heart holocytochrome c. This original approach offers the unique opportunity of selective modifications both in the C-terminal and in the N-terminal regions of the apoprotein and may represent an useful alternative to site-directed mutagenesis, particularly when D-amino acids, chemically and/or isotopically modified or other unnatural amino acids have to be introduced...
Borin G, Filippi B, Stivanello D, Marchiori F.A solution synthesis of Z-Gly-Thr-Lys (Tfa)-Met-Ile-Phe-Ala-Gly-Ile-Lys (Tfa)-Lys (Tfa)-NHNH-Boc corresponding to the sequence 77-87 of horse heart cytochrome c is described. The protected undecapeptide was obtained from intermediate hepta- and tetrapeptide fragments by an azide coupling.
Yamada S, Suruga K, Ogawa M, Hama T, Satoh T, Kawachi R, Nishio T, Oku T.The appearance of NO2- reducing activity of cytochrome c (Cyt c) upon heat denaturation was investigated with equine heart Cyt c. Denatured equine heart Cyt c (dCyt c), which was treated at 100 degrees C for 30 min, had NO2- reducing activity in the presence of dithionite and methylviologen in an aqueous solution under anaerobic conditions. In contrast, hemoglobin and myoglobin had no such activity under the same conditions. Using spectroscopic methods, we found that the appearance of this activity in the Cyt c was due to the following intramolecular changes: unfolding of the peptide chain, ex...
Semevolos SA, Marchant EA.The marginal transitional zone (MTZ) is peripherally located within the diarthrodial joint, and represents the junction of synovium, fibrous joint capsule, articular cartilage, periosteum, and bone. The purpose of this study is to characterize age-related differences in protein expression of matrix and molecular regulators in the marginal transitional zone of neonatal and weanling foals. Several families of proteins with known roles in cartilage and bone development are investigated, including matrix molecules, members of the Wnt signaling family, apoptotic factors and paracrine cell signaling...
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